*Target (protein/gene name): E3 ubiquitin-protein ligase/sspH1 *NCBI Gene # or RefSeq#: 888858 [NCBI] RefSeq#: YP_005237063.1.NC_016856.1. *Protein ID (NP or XP #) or Wolbachia#: NC_020282.1 *Organism (including strain): Salmonella typhimurium (strain 14028s / SGSC 2262)
Etiologic Risk Group (see link below): Risk group lv 2 (moderate individual risk, low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Many types of Salmonella bacteria cause salmonellosis in animals and people. While the occurrence of different types of Salmonella varies from country to country, S. typhimurium and S. enteritidis are the two most commonly found in the United States.
Some strains of Salmonella have become resistant to several antibiotics normally used to treat people with salmonella disease, posing a serious public health threat.
Every year, Salmonella is estimated to cause one million illnesses in the United States, with 19,000 hospitalizations and 380 deaths. Most persons infected with Salmonella develop diarrhea, fever, and abdominal cramps 12 to 72 hours after infection.
The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons, the diarrhea may be so severe that the patient needs to be hospitalized.
Link to TDR Targets page (if present): Not available
Essentiality of this protein: The protein interferes with the host's ubiquitination pathway and targets host proteins for proteasomal degradation
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives ---- Chain used for homology:
Current Inhibitors: paH enzymes Expression Information (has it been expressed in bacterial cells): Yersinia pestis Purification Method:
Protein constructs were expressed in BL21 (DE3) CodonPlus and induced with 0.3 mm isopropyl-1-thio-β-d-galactopyranoside at 16 °C for overnight. Expressed proteins were purified using nickel-nitrilotriacetic acid affinity chromatography and stored in a buffer containing 20 mmHEPES (pH 7.5), 150 mm NaCl, 5% glycerol, and 1 mm DTT. Purification tags on UBE1, UBE2D3, and ubiquitin were removed by tobacco etch virus protease. Image of protein (PyMol with features delineated and shown separately):
Crystal structure of SspH1 LRR domain in complex PKN1 HR1b domain
*length of your protein in Amino Acids: 700 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 77,119 Molar Extinction coefficient of your protein at 280 nm wavelength: 91370 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 888858 [NCBI] RefSeq#: YP_005237063.1.NC_016856.1.
*Protein ID (NP or XP #) or Wolbachia#: NC_020282.1
*Organism (including strain): Salmonella typhimurium (strain 14028s / SGSC 2262)
Etiologic Risk Group (see link below): Risk group lv 2 (moderate individual risk, low community risk)
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Many types of Salmonella bacteria cause salmonellosis in animals and people. While the occurrence of different types of Salmonella varies from country to country, S. typhimurium and S. enteritidis are the two most commonly found in the United States.
Some strains of Salmonella have become resistant to several antibiotics normally used to treat people with salmonella disease, posing a serious public health threat.
Every year, Salmonella is estimated to cause one million illnesses in the United States, with 19,000 hospitalizations and 380 deaths. Most persons infected with Salmonella develop diarrhea, fever, and abdominal cramps 12 to 72 hours after infection.
The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons, the diarrhea may be so severe that the patient needs to be hospitalized.
Link to TDR Targets page (if present): Not available
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/gene/14612615
Essentiality of this protein: The protein interferes with the host's ubiquitination pathway and targets host proteins for proteasomal degradation
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 6.3.2.19
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.19
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below):
http://www.sigmaaldrich.com/catalog/search?term=E3+ubiquitin-protein+ligase%2FsspH1&interface=All&N=0&mode=match%20partialmax&lang=en®ion=US&focus=product
-- -or link (or citation) to paper that contains assay information:
http://www.sciencedirect.com/science/article/pii/S1369527409001799
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: paH enzymes
Expression Information (has it been expressed in bacterial cells): Yersinia pestis
Purification Method:
Protein constructs were expressed in BL21 (DE3) CodonPlus and induced with 0.3 mm isopropyl-1-thio-β-d-galactopyranoside at 16 °C for overnight. Expressed proteins were purified using nickel-nitrilotriacetic acid affinity chromatography and stored in a buffer containing 20 mmHEPES (pH 7.5), 150 mm NaCl, 5% glycerol, and 1 mm DTT. Purification tags on UBE1, UBE2D3, and ubiquitin were removed by tobacco etch virus protease.
Image of protein (PyMol with features delineated and shown separately):
http://www.rcsb.org/pdb/explore/explore.do?structureId=4NKG
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MFNIRNTQPS VSMQAIAGAA APEASPEEIV WEKIQVFFPQ ENYEEAQQCL
AELCHPARGM LPDHISSQFA RLKALTFPAW EENIQCNRDG INQFCILDAG
SKEILSITLD DAGNYTVNCQ GYSEAHDFIM DTEPGEECTE FAEGASGTSL
RPATTVSQKA AEYDAVWSKW ERDAPAGESP GRAAVVQEMR DCLNNGNPVL
NVGASGLTTL PDRLPPHITT LVIPDNNLTS LPELPEGLRE LEVSGNLQLT
SLPSLPQGLQ KLWAYNNWLA SLPTLPPGLG DLAVSNNQLT SLPEMPPALR
ELRVSGNNLT SLPALPSGLQ KLWAYNNRLT SLPEMSPGLQ ELDVSHNQLT
RLPQSLTGLS SAARVYLDGN PLSVRTLQAL RDIIGHSGIR IHFDMAGPSV
PREARALHLA VADWLTSARE GEAAQADRWQ AFGLEDNAAA FSLVLDRLRE
TENFKKDAGF KAQISSWLTQ LAEDAALRAK TFAMATEATS TCEDRVTHAL
HQMNNVQLVH NAEKGEYDNN LQGLVSTGRE MFRLATLEQI AREKAGTLAL
VDDVEVYLAF QNKLKESLEL TSVTSEMRFF DVSGVTVSDL QAAELQVKTA
ENSGFSKWIL QWGPLHSVLE RKVPERFNAL REKQISDYED TYRKLYDEVL
KSSGLVDDTD AERTIGVSAM DSAKKEFLDG LRALVDEVLG SYLTARWRLN
*length of your protein in Amino Acids: 700
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 77,119
Molar Extinction coefficient of your protein at 280 nm wavelength: 91370
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):